1. Field of the Invention
The present invention relates to an immersion microscope objective lens suitable for observing an object (specimen) filled with a liquid, and particularly to an immersion microscope objective lens suitable for a total internal reflection fluorescence microscope (TIRFM).
2. Related Background Art
An immersion observation of an object (specimen) is a method to observe the object with a high magnification by filling an optical path between an objective lens and the object to be observed with a liquid such as water or oil to make a numerical aperture NA of the immersion microscope objective lens larger than one (>1) in accordance with refractive index of the liquid.
A total internal reflection fluorescence microscope using an immersion microscope objective lens has been known (see Japanese Patent Application Laid-Open No. 2003-15046). This has shown an observation method of an object to be observed by illuminating the object with evanescent light oozing out to the object side through a cover glass, an immersion liquid, and an immersion microscope objective lens and collecting fluorescence from the object. The evanescent light is weak light produced when illumination light is reflected with total internal reflection from a boundary surface between an object and a cover glass. In order to produce the evanescent light, it is necessary that the illumination light is incident to the boundary surface between an object and a cover glass with a large angle (more than the critical angle), so that an immersion microscope objective lens with a large numerical aperture NA is indispensable. According to the total internal reflection fluorescence microscope, information of the object only near the boundary surface can be effectively obtained.
To make the numerical aperture NA of an immersion microscope objective lens large, it is generally desired that refractive index of the immersion liquid is increased. Incidentally, as to refractive index at d-line (λ=587 nm), water is 1.333, glycerin is 1.473, and oil is 1.515. Accordingly, when oil with highest refractive index among them is used for an immersion liquid, the numerical aperture NA of an immersion microscope objective lens can be made large. In order to make the numerical aperture NA further large, a special oil having higher refractive index than the oil (refractive index is 1.515) has been developed. For example, Japanese Patent Application Laid-Open No. 2003-15046 discloses di-phenyl ether with refractive index of about 1.579.
However, a special oil with high refractive index usually has a lot of additives. Accordingly, when an object is observed with a fluorescence observation using a special oil as an immersion liquid, noise component caused by autofluorescence of the additives in the special oil becomes large. Although an oil with the least noise component is the above-described ordinary oil with refractive index of 1.515 (at d-line, 23° C.), the oil does not provide a sufficient numerical aperture NA. As long as using an ordinary oil, the largest numerical aperture NA of an immersion microscope objective lens has been 1.45 so far.